Diagnostic and therapeutic uses of gelsolin in renal failure

ABSTRACT

The invention is directed, in part, to the use of gelsolin to diagnose, monitor, and treat subjects with renal failure (e.g., chronic renal failure subjects on dialysis).

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/358,868, filed Jan. 23, 2009, which application claims the benefitunder 35 U.S.C. § 119(e) of United States provisional application Ser.No. 61/023,789, filed Jan. 25, 2008, the entire contents of which areincorporated by reference herein.

FEDERALLY SPONSORED RESEARCH

Some aspects of the present invention were made with support by grantsfrom the United States National Institutes of Health (NIH) under NIHgrants DK 71674 and DK 67397. The U.S. Government has certain rights inthe invention.

FIELD OF INVENTION

The invention is directed to diagnostic and therapeutic uses ofgelsolin.

BACKGROUND OF THE INVENTION

Chronic hemodialysis (HD) has drastically reduced the acute mortality ofend-stage renal disease (ESRD). Nevertheless, chronic renal failurepatients undergoing HD still die at a markedly accelerated rate. Thisadverse outcome appears early, with death ensuing far faster than inage-matched control populations within a year of initiating dialysis,and the most frequent causes of death are cardiovascular events andacute infections¹⁻³. Patients with chronic renal failure exhibitmanifestations of diffuse tissue injury, chronic inflammation, loss ofmuscle mass and hypoalbuminemia, and severe malnutrition, and all havebeen strongly linked with adverse outcomes⁴⁻⁸. The pathogenesismediating the connection between the aggregate of these underlyingconditions and accelerated mortality is largely unknown. Thus, thesearch for novel biomarkers that can reliably identify those ESRD and/orHD patients at increased risk of early death, and especially thosebiomarkers that are linked to potential therapies, may have significantclinical impact in improving the outcomes of this otherwise unfortunatepopulation.

SUMMARY OF THE INVENTION

Plasma gelslolin (pGSN) is a sensitive marker of tissue injury withstrong links to nutritional status, inflammation, and muscle mass, andimportantly, with possible therapeutic utility⁹⁻¹². pGSN is theextracellular variant of a protein, encoded on human chromosome 9, withcellular and secreted isoforms deployed by alternative mRNA splicing¹³.Cellular gelsolin (cGSN) is a widely expressed mediator of cell shapechange and motility through its regulated actin filament bindingfunction¹⁴. Plasma gelsolin is an abundant plasma protein thatcirculates in healthy individuals at an average concentration of 250 mg/L¹⁴. cGSN and pGSN are identical in primary structure and with respectto biochemical functions in vitro, except that pGSN contains anadditional 25 amino acids at its amino terminal and has a processedsignal sequence responsible for its secretion¹³. Many cell types secretepGSN, although as the bulkiest body organ, striated muscle, accounts formost pGSN production. The amino acid sequence of pGSN is highlyconserved between species, and no human anti-pGSN antibodies have beendescribed¹⁵.

Diverse conditions associated with acute tissue injury result inreductions in the circulating concentration of pGSN, the diminution inlevels is proportional to the degree of injury, and critical extents ofpGSN reduction are associated with adverse outcomes includingdeath^(11, 14, 16-18). Although exposure of cytoplasmic actin to theextracellular environment due to membrane disruption in tissue andendothelial injury is likely the mechanism of pGSN depletion^(19, 20),pGSN depletion also results from buffering a variety of circulatinginflammatory mediators (e.g., platelet activating factor,lysophosphatidic acid, lipopolysaccharide)^(12, 21) that potentiallymediate adverse complications.

This invention is based, in part, on the discovery that, in chronicdialysis subjects, baseline plasma gelsolin levels are low and thatgelsolin levels are inversely related to mortality risk such as frominfectious causes or cardiac causes. Thus, the invention involves, inone aspect, using gelsolin to characterize a renal failure subject'smortality risk and to monitor the efficacy of therapy. The invention isalso based on the discovery that, in chronic dialysis subjects, elevatedplasma actin levels are related to mortality risk. Thus, the inventioninvolves, in one aspect, using actin to characterize a renal failuresubject's mortality risk and to monitor the efficacy of therapy. Onecorrelate of these observations is that monitoring of plasma gelsolinlevels and/or actin levels could become part of the management strategyof renal failure.

According to one aspect of the invention, a method for characterizing arenal failure subject's mortality risk is provided. The method comprisescomparing a level of gelsolin from the subject to a predetermined value,and characterizing the subject's mortality risk based upon the level ofgelsolin in comparison to the predetermined value. A level of gelsolinbelow the predetermined value indicates the subject has an increasedmortality risk. In some embodiments, the predetermined value is about190 nanograms/microliter (ng/μl) of plasma. In some embodiments, thepredetermined value is about 150 ng/μl of plasma. In some otherembodiments, the predetermined value is about 120 ng/μl of plasma. Insome embodiments, a lower level of gelsolin indicates that the subjecthas a higher mortality risk. The method may further comprise obtainingthe level of gelsolin from the subject.

According to another aspect of the invention, a method forcharacterizing a renal failure subject's mortality risk is provided. Themethod comprises comparing a level of actin from the subject to apredetermined value and characterizing the subject's mortality riskbased upon the level of actin in comparison to the predetermined value.A level of actin above the predetermined value indicates the subject hasan increased mortality risk. In some embodiments, the predeterminedvalue is about 0.01 micrograms/milliliter (μg/ml ) of plasma. In someembodiments, the predetermined value is about 0.1 μg/ml of plasma. Insome embodiments, a higher level of actin correlates with a highermortality risk. The method may further comprise obtaining the level ofactin from the subject.

According to yet another aspect of the invention, a method forcharacterizing a renal failure subject's mortality risk is provided. Themethod comprises comparing a level of gelsolin from the subject to afirst predetermined value to establish a first risk value and comparinga level of actin from the subject to a second predetermined value toestablish a second risk value. The subject's mortality risk ischaracterized based upon the combination of the first risk value and thesecond risk value wherein the combination of the first risk value andsecond risk value establishes a third risk value different from saidfirst and second risk values. In some embodiments, the firstpredetermined value is about 190 ng/μl of plasma. In some embodiments,the first predetermined value is about 150 ng/μl of plasma. In otherembodiments, the first predetermined value is about 120 ng/μl of plasma.In some embodiments, the second predetermined value is about 0.01 μg/mlof plasma. In some embodiments, the predetermined value is about 0.1μg/ml of plasma.

The method may further involve obtaining the level of gelsolin from thesubject. In some embodiments, the method may further comprise obtainingthe level of actin from the subject.

According to yet another aspect of the invention, a method forevaluating the efficacy of a therapy in a renal failure subject isprovided. The method involves comparing a level of gelsolin from thesubject to a predetermined value and determining whether the level ofgelsolin is at or above the predetermined level said determination beingindicative that the therapy is efficacious.

The steps of the method may be repeated so as to monitor the subject'slevels of gelsolin over time. In some embodiments, the predeterminedvalue is about 190 ng/μl of plasma. In some embodiments, thepredetermined value is about 150 ng/μl of plasma. In other embodiments,the predetermined value is about 120 ng/μl of plasma. According to yetanother aspect of the invention, a method for treating a renal failuresubject is provided. The method involves administering an effectiveamount of gelsolin to a subject in need of such a treatment to raise thelevel of gelsolin in the subject above a predetermined value. In someembodiments, the predetermined value is about 190 ng/μl of plasma. Insome embodiments, the predetermined value is about 150 ng/μl of plasma.In other embodiments, the predetermined value is about 120 ng/μl ofplasma.

The gelsolin may be plasma gelsolin (pGSN), cytoplasmic gelsolin (cGSN),advillin, villin, capG, flightless proteins, fragmin, severin,adseverin, protovillin, and/or supervillin. The gelsolin may beadministered orally, sublingually, buccally, intranasally,intravenously, intramuscularly, intrathecally, intraperitoneally, orsubcutaneously.

According to still another aspect of the invention, a method fortreating a renal failure subject having or at risk of developing aninfection is provided. The method involves administering gelsolin to asubject in need of such a treatment in an effective amount to reduce therisk of the infection.

According to yet another aspect of the invention, a method of treatmentto raise the level of gelsolin in a renal failure subject is provided.The method comprises instructing the renal failure subject in need ofsuch a treatment to take an effective amount of gelsolin for the purposeof raising the level of gelsolin in the subject above a predeterminedvalue. In some embodiments, the predetermined value is about 190 ng/μlof plasma. In some embodiments, the predetermined value is about 150ng/μl of plasma. In other embodiments, the predetermined value is about120 ng/μl of plasma.

The gelsolin may be plasma gelsolin (pGSN), cytoplasmic gelsolin (cGSN),advillin, villin, capG, flightless proteins, fragmin, severin,adseverin, protovillin, and/or supervillin. The gelsolin may beadministered orally, sublingually, buccally, intranasally,intravenously, intramuscularly, intrathecally, intraperitoneally, orsubcutaneously.

According to still another aspect of the invention, a method fortreating a renal failure subject to raise the level of gelsolin in thesubject is provided. The method comprises providing the subject with apackage containing gelsolin, and providing the subject with indiciaindicating that the gelsolin is for raising the level of gelsolin in thesubject above a predetermined value. In some embodiments, thepredetermined value is about 190 ng/μl of plasma. In some embodiments,the predetermined value is about 150 ng/μl of plasma. In otherembodiments, the predetermined value is about 120 ng/μl of plasma.

The gelsolin may be plasma gelsolin (pGSN), cytoplasmic gelsolin (cGSN),advillin, villin, capG, flightless proteins, fragmin, severin,adseverin, protovillin, and/or supervillin. The gelsolin may beadministered orally, sublingually, buccally, intranasally,intravenously, intramuscularly, intrathecally, intraperitoneally, orsubcutaneously.

According to another aspect of the invention, a medical treatmentproduct is provided. The product comprises a package containing gelsolinand indicia indicating that the gelsolin is for raising the level ofgelsolin in a renal failure subject above a predetermined value. In someembodiments, the predetermined value is about 190 ng/μl of plasma. Insome embodiments, the predetermined value is about 150 ng/μl of plasma.In other embodiments, the predetermined value is about 120 ng/μl ofplasma.

The gelsolin may be plasma gelsolin (pGSN), cytoplasmic gelsolin (cGSN),advillin, villin, capG, flightless proteins, fragmin, severin,adseverin, protovillin, and/or supervillin. The gelsolin may beadministered orally, sublingually, buccally, intranasally,intravenously, intramuscularly, intrathecally, intraperitoneally, orsubcutaneously.

According to yet another aspect of the invention, the use of gelsolin inthe manufacture of a medicament for raising the level of gelsolin in arenal failure subject above a predetermined value is provided. Thegelsolin may be plasma gelsolin (pGSN), cytoplasmic gelsolin (cGSN),advillin, villin, capG, flightless proteins, fragmin, severin,adseverin, protovillin, and/or supervillin. The gelsolin may beadministered orally, sublingually, buccally, intranasally,intravenously, intramuscularly, intrathecally, intraperitoneally, orsubcutaneously.

The invention also provides for a method that comprises comparing alevel of gelsolin in a renal failure subject to a predetermined valueand, if the level of gelsolin is below the predetermined value,identifying the subject as having an increased mortality risk. In someembodiments, the method comprises advising the subject about the risk,treatment, or medical care. In some embodiments, the treatment comprisesgelsolin. In some embodiments, the method comprises obtaining the levelof gelsolin form the subject. In some embodiments, the predeterminedvalue is about 190 ng/μl of plasma. In some embodiments, thepredetermined value is about 150 ng/μl of plasma. In other embodiments,the predetermined value is about 120 ng/μl of plasma.

According to another aspect, the invention provides a method thatcomprises comparing a level of actin in a renal failure subject to apredetermined value and, if the level of actin is above thepredetermined value, identifying the subject as having an increasedmortality risk. In some embodiments, the method comprises advising thesubject about the risk, treatment, or medical care. In some embodiments,the treatment comprises gelsolin. In some embodiments, the methodcomprises obtaining the level of actin form the subject. In someembodiments, the predetermined value is about 0.01 μg/ml of plasma. Insome embodiments, the predetermined value is about 0.1 μg/ml of plasma.

According to yet another aspect, the invention provides a method thatcomprises performing an assay to detect a level of gelsolin in a renalfailure subject, wherein the assay comprises a predetermined value thatpredicts increased mortality risk in the subject. In some embodiments,the method comprises advising the subject about the risk, treatment, ormedical care. In some embodiments, the treatment comprises gelsolin. Insome embodiments, the predetermined value is about 190 ng/μl of plasma.In some embodiments, the predetermined value is about 150 ng/μl ofplasma. In other embodiments, the predetermined value is about 120 ng/μlof plasma.

According to still another aspect, the invention provides a method thatcomprises performing an assay to detect a level of actin in a renalfailure subject, wherein the assay comprises a predetermined value thatpredicts increased mortality risk in the subject. In some embodiments,the method comprises advising the subject about the risk, treatment, ormedical care. In some embodiments, the treatment comprises gelsolin. Insome embodiments, the predetermined value is about 0.01 μg/ml of plasma.In some embodiments, the predetermined value is about 0.1 μg/ml ofplasma.

The following embodiments apply to various aspects of the invention setforth herein unless indicated otherwise.

The level of gelsolin or actin may be in a body fluid of the subject.Examples of body fluids include but are not limited to blood, plasma,serum, urine, synovial fluid, cerebrospinal or alveolar fluid. In someimportant embodiments, the body fluid is plasma.

In some embodiments, the mortality is caused by an infection. Theinfection may be caused by a gram-positive bacterium, a gram-negativebacterium, an acid-fast bacillus, a spirochete, an actinomycete, avirus, a fungus, a parasite, Ureoplasma species, Mycoplasma species,Chlamydia species, or Pneumocystis species.

Examples of gram-positive bacteria include but are not limited toPasteurella species, Staphylococcus species, Streptococcus species,Bacillus anthracis, Corynebacterium species, Diphtheroids species,Listeria species, Erysipelothrix species, and Clostridium species.

Examples of gram-negative bacteria include but are not limited toNeisseria species, Branhamella species, Escherichia species,Enterobacter species, Proteus species, Pseudomonas species, Klebsiellaspecies, Salmonella species, Shigella species, Serratia species,Acinetobacter species, Haemophilus species, Brucella species, Yersiniaspecies, Francisella species, Pasturella species, Vibrio choleraspecies, Flavobacterium species, Pseudomonas species, Campylobacterspecies, Bacteroides species, Fusobacterium species, Calymmatobacteriumspecies, Streptobacillus species, and Legionella species.

The acid-fast bacillus may be a Mycobacterium species. The spirochetemay be Treponema species, Borrelia species, or Leptospira species.

Examples of viruses include but are not limited to Retro viruses, humanimmunodeficiency viruses, Cytomegaloviruses, Picorna viruses, Polioviruses, hepatitis A virus, enteroviruses, Coxsackie viruses,rhinoviruses, echoviruses, Calciviruses, Toga viruses, equineencephalitis viruses, rubella viruses, Flaviviruses, dengue viruses,encephalitis viruses, yellow fever viruses, coronaviruses,Rhabdoviruses, vesicular stomatitis viruses, rabies viruses,Filoviruses, ebola virus, Paramyxo viruses, parainfluenza viruses, mumpsvirus, measles virus, respiratory syncytial virus, Orthomyxoviruses,influenza viruses, Hantaan viruses, bunga viruses, phleboviruses, Nairoviruses, Arena viruses, hemorrhagic fever viruses, reoviruses,orbiviruses, rotaviruses, Birnaviruses, Hepadnaviruses, Hepatitis Bvirus, parvoviruses, Papovaviruses, papilloma viruses, polyoma viruses,Adenoviruses, Herpes viruses, varicella zoster virus, Pox viruses,variola viruses, vaccinia viruses, Iridoviruses, African swine feverviruses, delta hepatitis virus, non-A, non-B hepatitis virus, HepatitisC, Norwalk viruses, astroviruses, and unclassified viruses.

Examples of fungi include but are not limited to Cryptococcus species,Histoplasma species, Coccidioides species, Paracoccidioides species,Blastomyces species, Chlamydia species, Candida species, Sporothrixspecies, Aspergillus species, and fungus of mucormycosis.

Examples of parasites include but are not limited to Plasmodium species,Toxoplasma species, Babesia species, Leishmania species, and Trypanosomaspecies.

In some embodiments, the mortality is caused by a cardiovascular event.The cardiovascular event may be acute coronary syndrome, myocardialinfarction, congestive heart failure, stroke, or sudden death.

In some embodiments, the subject is on dialysis. The dialysis may behemodialysis or peritoneal dialysis. In some embodiments, the subjecthas end-stage renal disease (ESRD).

In some embodiments, the subject is otherwise free of indicationscalling for treatment. A subject free of indications calling fortreatment with gelsolin is a subject who has no signs or symptomscalling for treatment with gelsolin. Gelsolin is indicated for thetreatment of septic shock. Gelsolin is also indicated for the treatmentof actin-related disorders such as Adult Respiratory Distress Syndrome(ARDS), fulminant hepatic necrosis, acute renal failure, muscle injury,disorders characterized by elevated levels of BUN and/or creatinine.Actin-related disorders are known to those of ordinary skill in the art.

In some embodiments, the first predetermined value may be a plurality ofpredetermined gelsolin level ranges, one of a plurality of ranges beingbelow about 190 ng/μl of plasma and another of said ranges being aboveabout 190 ng/μl of plasma, and the comparing step comprises determiningin which of said plurality of predetermined gelsolin level ranges saidsubject's gelsolin level falls.

In some embodiments, the first predetermined value may be a plurality ofpredetermined gelsolin level ranges, one of a plurality of ranges beingbelow about 150 ng/μl of plasma and another of said ranges being aboveabout 150 ng/μl of plasma, and the comparing step comprises determiningin which of said plurality of predetermined gelsolin level ranges saidsubject's gelsolin level falls. In some embodiments, the firstpredetermined value may be a plurality of predetermined gelsolin levelranges, one of a plurality of ranges being below about 120 ng/μl ofplasma and another of said ranges being above about 120 ng/μl of plasma.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention. The invention is capable of other embodiments and of beingpracticed or of being carried out in various ways. Also, the phraseologyand terminology used herein is for the purpose of description and shouldnot be regarded as limiting. The use of “including”, “comprising”, or“having”, “containing”, “involving”, and variations thereof herein, ismeant to encompass the items listed thereafter and equivalents thereofas well as additional items.

These and other aspects of the inventions, as well as various advantagesand utilities will be apparent with reference to the DetailedDescription of the Invention. Each aspect of the invention can encompassvarious embodiments as will be understood.

All documents identified in this application are incorporated in theirentirety herein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a histogram showing the distribution of plasma gelsolin in arandom sampling of 150 ESRD subjects from throughout the United States.The dashed line represents the mean pGSN level of 250 mg/L in healthycontrols.

FIG. 2 are two graphs showing the 1-year survival among chronic dialysispatients according to indicated median plasma gelsolin level (top panel)or according to baseline levels of plasma gelsolin subdivided intoindicated tertiles (bottom panel). Kaplan Meier analysis of one-yearsurvival according to baseline pGSN levels categorized by the medianlevel (top panel; log-rank p =0.05) and tertiles (Bottom panel; log-rankp =0.02.) in the controls.

FIG. 3 is a picture of a Western blot detecting plasma actin fromsubjects with indicated pGSN tertiles.

FIG. 4 is a histogram showing the risk of 1-year mortality according tobaseline plasma gelsolin and actin levels. Compared to those withelevated baseline pGSN (+pGSN) and no detectable actin (−actin), themultivariable risk of one-year death among those with low baseline pGSN(−pGSN) and detectable actin (+actin) was significantly elevated: −pGSN,+actin, OR 9.8, 95% CI 2.9-33.5; +pGSN, +actin, OR 3.6, 95% CI 1.0-13.5;−pGSN, −actin, OR 1.6, 95% CI 0.3-7.7; +pGSN, −actin, OR 1.0 (ref). * p=0.05, ** p =0.01.

FIG. 5 is a graph showing the correlation between baseline plasmagelsolin (pGSN) levels and estimated glomerular filtration rate in 53patients with chronic kidney disease.

FIG. 6 is a histogram showing median pGSN levels in patients withchronic kidney disease. Stage 1 (n=5), estimated glomerular filtrationrate (eGFR)>90 ml/min/1.73 m²; Stage 2 (n=11), eGFR 60-89 ml/min/1.73m²; Stage 3 (n=18), eGFR 30-59 ml/min/1.73 m²; Stage 4 (n=19), eGFR15-29 ml/min/1.73 m². Error bars represent inter-quartile ranges.

FIG. 7 is a picture of some possible mechanisms for pGSN depletion andits consequences in chronic renal failure. Chronic renal failureinhibits pGSN synthesis and accelerates clearance. Muscle is the majorsource of pGSN biosynthesis, and the reduction of muscle mass associatedwith chronic renal failure would reduce net pGSN production. The failureto eliminate toxins in renal failure causes widespread tissue,especially endothelial, destruction leading to exposure of cytoplasmicactin into the plasma and pGSN sequestration in broken cells. Inaddition, release of inside-out membrane vesicles with attached actinfilaments from damaged cells would result in detectable circulatingactin, and circulating actin accelerates pGSN clearance. Low pGSNresults in impaired buffering of inflammatory mediators such asplatelet-activating factor, promoting vascular complications, andrendering patients susceptible to the lethal effects of sepsis.

DETAILED DESCRIPTION

This invention is based, in part, on the discovery that, in chronicdialysis subjects, baseline plasma gelsolin levels are low and gelsolinlevels are inversely related to mortality (e.g., mortality frominfectious or cardiac causes). The invention is also based, in part, onthe discovery that, in chronic dialysis subjects, elevated plasma actinlevels are directly related to mortality. Therefore, gelsolin depletionand/or excess actin predict mortality in renal failure subjects (e.g.,chronic dialysis subjects). It is believed that gelsolin may be used toreduce mortality and/or to reduce the risk of infection in a renalfailure subject (e.g., chronic dialysis subject).

Thus, the invention involves, in some aspects, administering gelsolin toa renal failure subject (e.g., chronic dialysis subject) to raise thelevel of gelsolin in the subject and to reduce mortality and/or toreduce the risk of infection in the renal failure subject (e.g., chronicdialysis subject). The term “treat” or “treatment” is intended toinclude prophylaxis, amelioration, prevention or cure from thecondition.

As used herein, the term “gelsolin” encompasses wild type gelsolin(GenBank accession No.: X04412), isoforms, analogs, variants, fragmentsor functional derivatives of gelsolin.

Gelsolin (GSN), unlike other mammalian proteins, has both cytoplasmic(cGSN) and secreted or exported isoforms, also called plasma gelsolin(pGSN), which are derived by alternative splicing of the message from asingle gene (Sun et al. J. Biol. Chem. 274:33179-33182 (1999)). As usedherein, gelsolin isoforms include versions of gelsolin with some smalldifferences in their amino acid sequences, usually a splice variant orthe result of some posttranslational modification.

Gelsolin encompasses native as well as synthetic and recombinantgelsolin. Gelsolin is an abundant secretory protein (Yin, H. L.,Kwiatkowski, D. J., Mole, J. E. & Cole, F. S. (1984) J Biol Chem 259,5271-6). The exported isoform of gelsolin, pGSN, has 25 additional aminoacids and originates from alternative splicing of a single gene(Kwiatkowski, D. J., Stossel, T. P., Orkin, S. H., Mole, J. E., Colten,H. R. & Yin, H. L. (1986) Nature 323, 455-8). Recombinant human gelsolin(rhGSN) (Biogen IDEC, Inc., Cambridge, Mass.) is produced in E. coli,and though it has the same primary structure as the native protein,under standard conditions of purification, it differs from natural humanplasma gelsolin by a disulfide bond that is present in the naturalprotein. The recombinant protein is, therefore, properly oxidized afterpurification, and its structure and functions are indistinguishable fromhuman plasma gelsolin (Wen et. al., Biochemistry 35:9700-9709 (1996)).In some of the therapeutic aspects and embodiments of the invention, theuse of rhGSN is preferred. In some of the diagnostic aspects andembodiments of the invention, the use of pGSN is preferred.

A “gelsolin analog” refers to a compound substantially similar infunction to either the native gelsolin or to a fragment thereof.Gelsolin analogs include biologically active amino acid sequencessubstantially similar to the gelsolin sequences and may havesubstituted, deleted, elongated, replaced, or otherwise modifiedsequences that possess bioactivity substantially similar to that ofgelsolin. For example, an analog of gelsolin is one which does not havethe same amino acid sequence as gelsolin but which is sufficientlyhomologous to gelsolin so as to retain the bioactivity of gelsolin.Bioactivity can be determined, for example, by assaying gelsolin'sability to stimulate actin nucleation. Gelsolin bioactivity assays areknown to those of ordinary skill in the art.

A gelsolin “variant” is meant to refer to a compound that issubstantially similar in structure and bioactivity either to nativegelsolin, or to a fragment thereof. The term variant encompasses thegelsolin family of proteins. The gelsolin family of proteins is a groupof actin binding proteins sharing repeats of about 15 kDa homologousdomains that adopt a similar fold. Examples gelsolin family proteinsinclude but are not limited to advillin, villin, capG, flightlessproteins, fragmin, severin, adseverin, protovillin, and supervillin.

A gelsolin “fragment” is meant to include any portion of a gelsolinmolecule which provides a segment of gelsolin which maintains thebioactivity of gelsolin; the term is meant to include gelsolin fragmentswhich are made from any source, such as, for example, fromnaturally-occurring peptide sequences, synthetic orchemically-synthesized peptide sequences, and genetically engineeredpeptide sequences.

A “functional derivative” of gelsolin is a derivative which possesses abioactivity that is substantially similar to the bioactivity ofgelsolin. By “substantially similar” is meant activity which isquantitatively different but qualitatively the same. For example, afunctional derivative of gelsolin could contain the same amino acidbackbone as gelsolin but also contains other modifications such aspost-translational modifications such as, for example, boundphospholipids, or covalently linked carbohydrate, depending on thenecessity of such modifications for the performance of the diagnosticassay or therapeutic treatment. As used herein, the term is also meantto include a chemical derivative of gelsolin. Such derivatives mayimprove gelsolin's solubility, absorption, biological half life, etc.The derivatives may also decrease the toxicity of gelsolin, or eliminateor attenuate any undesirable side effect of gelsolin, etc. Chemicalmoieties capable of mediating such effects are disclosed in Remington'sPharmaceutical Sciences (1980). Procedures for coupling such moieties toa molecule such as gelsolin are well known in the art. The term“functional derivative” is intended to include the “fragments,”“variants,” “analogues,” or “chemical derivatives” of gelsolin.

The invention involves in some aspects, methods for treating a renalfailure subject (e.g., chronic dialysis subject). The gelsolin isadministered in an amount effective to raise the level of gelsolin andor to reduce the level of actin in the subject.

A response to a treatment method of the invention can be determined, forexample, by measuring plasma or blood gelsolin and/or plasma or bloodactin to determine whether plasma or blood gelsolin levels are increasedand/or plasma or blood actin levels are decreased as a result of thetreatment. Tests and methods for measuring plasma or blood gelsolinand/or actin and interpreting results of such tests are known to thoseof ordinary skill in the art.

In another aspect of the invention, a method for monitoring therapy in asubject is provided. The method involves obtaining a level of gelsolinand/or a level of actin in a subject undergoing therapy. The level ofgelsolin is compared to a predetermined value corresponding to a controllevel of gelsolin (e.g., in an apparently healthy population). The levelof actin is compared to a predetermined value corresponding to a controllevel of actin (e.g., in an apparently healthy population). Adetermination of whether the level of gelsolin and/or actin is at, belowor above a predetermined level will indicate of whether the subjectwould benefit from continued therapy with the same therapy or wouldbenefit from a change in therapy. For example, in some embodiments, adetermination that the level of gelsolin is at or above a predeterminedlevel and/or the level of actin is at or below a predetermined levelwill indicate that the subject would benefit from continued therapy withthe same therapy. In some embodiments, a determination that the level ofgelsolin is at or below a predetermined level and/or the level of actinis at or above a predetermined level indicates that the subject wouldbenefit from change in therapy. In some embodiments, obtaining a levelof gelsolin and/or actin is repeated so as to monitor the subject'slevels of gelsolin and/or actin over time.

A change in therapy with gelsolin refers to an increase in the dose ofthe gelsolin, a switch from one gelsolin to another gelsolin, a switchfrom gelsolin to another agent, the addition of another agent to thegelsolin therapeutic regimen, or a combination thereof.

According to another aspect of the invention, a method for evaluatingthe efficacy of a therapy of renal failure in a subject is provided. Themethod comprises comparing a level of gelsolin to a predetermined valueand determining whether the level of gelsolin is at or above apredetermined level said determination being indicative that the therapyis efficacious. In some embodiments, the method comprises comparing alevel of actin to a predetermined value and determining whether thelevel of actin is at or below a predetermined level said determinationbeing indicative that the therapy is efficacious.

In some embodiments, the subject may have been undergoing the therapyfor at least 1, 2, 3, 4, 5, 6, 7 days or more. In some embodiments, thesubject may have been undergoing the therapy for at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12 weeks or more. In some embodiments, the subjectmay have been undergoing the therapy for at least 3, 4, 5, 6, 7, 8, 9,10, 11, 12 months or more.

One aspect of the invention is directed to the measurement of gelsolinlevels and/or actin levels to guide treatments in order to improveoutcome in subjects. Levels of gelsolin and/or actin have predictivevalue for response to treatments to reduce the risk of mortality in arenal failure subject. Subjects who would benefit from this aspect ofthis invention are renal failure subjects who are undergoing therapy toreduce the risk of mortality (e.g., from infections or cardiac causes)and to raise the level of gelsolin. A subject on-therapy is a subjectwho already has been diagnosed with renal failure (e.g., a subject onchronic hemodialysis) and is in the course of treatment with a therapy.The therapy can be any of the therapeutic agents used in the treatmentof renal failure. Therapeutic agents used in the treatment of renalfailure are known to those of ordinary skill in the art. The therapyalso can be non-drug treatments. In important embodiments, the therapyis one which increases levels of gelsolin and/or decreases levels ofactin. In some embodiments, the therapy is a therapy with gelsolin. Thesubject most likely to benefit from this invention is a human subjecton-therapy (e.g., a human subject with renal failure on therapy forrenal failure) and who has a gelsolin level at or below about 190 ng/μl(ng/μl) of plasma or who has an actin level at or above about 0.01 μg/mlof plasma. In some embodiments, the human subject on-therapy has agelsolin level at or below about 150 ng/μl of plasma. In someembodiments, the human subject on-therapy has a gelsolin level at orbelow about 120 ng/μl of plasma. In some embodiments, the human subjecton-therapy has an actin level at or above about 0.1 μg/ml of plasma.

In some embodiments, the subject already has or had an infection. Asubject who has or has had a primary (first) bacterial, viral, fungal,parasitic, or protozoal infection may be at an elevated risk of asecondary (second) infection. In some embodiments, the subject has nothad a primary infection, but is at an elevated risk of having aninfection because the subject has one or more risk factors to have aninfection. Examples of risk factors for a primary infection include:immunosuppression, immunocompromise, age, trauma, burns (e.g., thermalburns), surgery, foreign bodies, and cancer. The degree of risk of aninfection depends on the multitude and the severity or the magnitude ofthe risk factors that the subject has. Risk charts and predictionalgorithms are available for assessing the risk of an infection in asubject based on the presence and severity of risk factors.

In some embodiments, the treatment is gelsolin. Gelsolin may beadministered alone, in a pharmaceutical composition or combined withother therapeutic regimens. Gelsolin and optionally other therapeuticagent(s) may be administered simultaneously or sequentially. When theother therapeutic agents are administered simultaneously they can beadministered in the same or separate formulations, but are administeredat the same time. The other therapeutic agents may be administeredsequentially with one another and with gelsolin when the administrationof the other therapeutic agents and the gelsolin is temporallyseparated. The separation in time between the administration of thesecompounds may be a matter of minutes or it may be longer.

In practicing certain methods of the present invention, a level ofgelsolin in a subject is obtained. This level then is compared to apredetermined value, wherein the level of gelsolin in comparison to thepredetermined value is indicative of the likelihood that the subjectwill benefit from continued therapy. The subject then can becharacterized in terms of the net benefit likely to be obtained from achange in therapy.

The level of the gelsolin for the subject can be obtained by any artrecognized method. Typically, the level is determined by measuring thelevel of gelsolin in a body fluid, for example, blood, serum, plasma,lymph, saliva, urine, and the like. The level can be determined byELISA, or other immunoassays or other conventional techniques fordetermining the presence of gelsolin. Conventional methods may includesending a sample(s) of a subject's body fluid to a commercial laboratoryfor measurement. Methods for measuring gelsolin are described herein.

The invention also involves comparing the level of gelsolin and/or thelevel of actin for the subject with a predetermined value. Thepredetermined value can take a variety of forms. It can be a singlecut-off value, such as a median or mean. It can be established basedupon comparative groups, such as, for example, where the risk in onedefined group is double the risk in another defined group. It can be arange, for example, where the tested population is divided equally (orunequally) into groups, such as a low-risk group, a medium-risk groupand a high-risk group, or into quartiles, the lowest quartile beingsubjects with the highest risk and the highest quartile being subjectswith the lowest risk, or into tertiles the lowest tertile being subjectswith the highest risk and the highest tertile being subjects with thelowest risk. The predetermined value may be a cut-off value which ispredetermined by the fact that a group having a gelsolin level less thanthe cut-off value demonstrates a statistically significant increase inthe mortality risk as compared to a comparative group. In someembodiments the comparative group is a group having a higher level ofgelsolin.

The predetermined value can depend upon the particular population ofsubjects selected. Accordingly, the predetermined values selected maytake into account the category in which a subject falls. Appropriateranges and categories can be selected with no more than routineexperimentation by those of ordinary skill in the art. The preferredbody fluids are plasma and blood. In some embodiments, the predeterminedvalue of gelsolin is about 190 ng/μl of plasma. In some embodiments, thepredetermined value of gelsolin is about 150 ng/μl of plasma. In someembodiments, the predetermined value of gelsolin is about 120 ng/μl ofplasma. The predetermined value will depend, of course, upon thecharacteristics of the subject population in which the subject lies. Incharacterizing risk, numerous predetermined values can be established.

Commercial sources which produce reagents for assays for gelsolin. Theseinclude, for example, Cytoskeleton (Denver, Colo.), Sigma (St. Louis,Mo.) and Calbiochem (San Diego, Calif.)

In practicing certain methods of the present invention, it is requiredto obtain a level of actin in a subject. This level then is compared toa predetermined value, wherein the level of actin in comparison to thepredetermined value is indicative of the likelihood that the subjectwill benefit from continued therapy. The subject then can becharacterized in terms of the net benefit likely to be obtained from achange in therapy.

The level of the actin for the subject can be obtained by any artrecognized method. Typically, the level is determined by measuring thelevel of actin in a body fluid, for example, blood, serum, plasma,lymph, saliva, urine, and the like. The level can be determined asdescribed in the Example below, or other assays or other conventionaltechniques for determining the presence of actin. Conventional methodsmay include sending a sample(s) of a subject's body fluid to acommercial laboratory for measurement.

The invention also involves comparing the level of actin for the subjectwith a predetermined value. The predetermined value can take a varietyof forms. It can be a single cut-off value, such as a median or mean. Itcan be established based upon comparative groups, such as, for example,where the risk in one defined group is double the risk in anotherdefined group. It can be a range, for example, where the testedpopulation is divided equally (or unequally) into groups, such as alow-risk group, a medium-risk group and a high-risk group, or intoquartiles, the lowest quartile being subjects with the lowest risk andthe highest quartile being subjects with the highest risk, or intotertiles the lowest tertile being subjects with the lowest risk and thehighest tertile being subjects with the highest risk. The predeterminedvalue may be a cut-off value which is predetermined by the fact that agroup having an actin level higher than the cut-off value demonstrates astatistically significant increase in mortality risk as compared to acomparative group.

The predetermined value can depend upon the particular population ofsubjects selected. Accordingly, the predetermined values selected maytake into account the category in which a subject falls. Appropriateranges and categories can be selected with no more than routineexperimentation by those of ordinary skill in the art. In someembodiments, the predetermined value of actin is about 0.01 μg/ml ofplasma. In some embodiments, the predetermined value of actin is about0.1 μg/ml of plasma. The predetermined value will depend, of course,upon the characteristics of the subject population in which the subjectlies. In characterizing risk, numerous predetermined values can beestablished.

The invention provides methods for determining whether a subject willbenefit from continued therapy or would benefit from a change intherapy. The benefit is typically a reduction in the signs and symptomsor complications of renal failure (e.g., infectious or cardiovascularcomplications). Signs, symptoms, manifestations and complications ofrenal failure are known to those of ordinary skill in the art.

These methods have important implications for patient treatment and alsofor the clinical development of new therapies. Determining whether asubject will benefit from continued therapy or would benefit from achange in therapy is clinically useful. One example of clinicalusefulness of the methods of this invention includes identifyingsubjects who are less likely or more likely to respond to a therapy. Themethods of the invention are also useful in predicting or determiningthat a subject would benefit from continued therapy or would benefitfrom a change in therapy. Health care practitioners select therapeuticregimens for treatment based upon the expected net benefit to thesubject. The net benefit is derived from the risk to benefit ratio. Thepresent invention permits the determination of whether a subject willbenefit from continued therapy or would benefit from a change intherapy, thereby aiding the physician in selecting a therapy.

Another example of clinical usefulness, in the case of human subjectsfor example, includes aiding clinical investigators in the selection forclinical trials of subjects with a high likelihood of obtaining a netbenefit. It is expected that clinical investigators now will use thepresent invention for determining entry criteria for clinical trials.

A subject who would benefit from continued therapy is a subject whoseon-therapy level of gelsolin reaches a certain predetermined value orwhose level of gelsolin is increasing. Predetermined values of gelsolinare described above. A subject who would benefit from a change intherapy is a subject whose on-therapy level of the gelsolin did notreach a certain predetermined value or whose on-therapy level ofgelsolin is not increasing.

A subject who would also benefit from continued therapy is a subjectwhose on-therapy level of actin reaches a certain predetermined value orwhose level of actin is decreasing. Predetermined values of actin aredescribed above. A subject who would benefit from a change in therapy isa subject whose on-therapy level of the actin did not reach a certainpredetermined value or whose on-therapy level of actin is notdecreasing.

As used herein, a “change in therapy” refers to an increase or decreasein the dose of the existing therapy, a switch from one therapy toanother therapy, an addition of another therapy to the existing therapy,or a combination thereof. A switch from one therapy to another mayinvolve a switch to a therapy with a high risk profile but where thelikelihood of expected benefit is increased. In some embodiments,preferred therapies are therapies that increase the level(s) of gelsolinand/or that decrease the level(s) of actin. A subject who would benefitfrom a change in therapy by increasing the dose of the existing therapyis a subject who, for example, was on the therapy but was not receivingthe maximum tolerated dose or the maximum allowed dose of the therapyand whose level of gelsolin and/or actin did not reach a certainpredetermined value. In such instances the dose of the existing therapyis increased until the level of gelsolin and/or actin reaches a certainpredetermined value. In some instances, the dose of the existing therapyis increased from the existing dose to a higher dose that is not themaximum tolerated dose nor the maximum allowed dose of the therapy. Inother instances, the dose is increased to the maximum tolerated or tothe maximum allowed dose of the therapy. A subject who would benefitfrom a change in therapy by decreasing the dose of the existing therapyis, for example, a subject whose on-therapy level of gelsolin and/oractin reaches or can reach a certain predetermined value with a lowerdose of the therapy.

A subject who would benefit from a switch from one therapy to anothertherapy is, for example, a subject who was on the maximum tolerated doseor the maximum allowed dose of the therapy and whose level of gelsolinand/or actin did not reach a certain predetermined value. Anotherexample is a subject was not on the maximum tolerated or the maximumallowed dose of the therapy but was determined by a health carepractitioner to more likely benefit from another therapy. Suchdeterminations are based, for example, on the development in the subjectof unwanted side effects on the initial therapy or a lack of response tothe initial therapy.

A subject who would benefit from a change in therapy by the addition ofanother therapy to the existing therapy is, for example, a subject whowas on a therapy but whose level of gelsolin and/or actin did not reacha certain predetermined value. In such instances, another therapy isadded to the existing therapy. The therapy that is added to the existingtherapy can have a different mechanism of action in increasing the levelof gelsolin and/or decreasing the level of actin than the existingtherapy. In some instances, a combination of the aforementioned changesin therapy may be used.

The invention also provides methods for determining the efficacy of atherapy. The efficacy is typically the efficacy of the therapy inincreasing the level of gelsolin and/or decreasing the level of actin.This is sometimes also referred to as a positive response or a favorableresponse. Efficacy can be determined by a gelsolin blood test(s) and/oractin blood test(s) to determine whether gelsolin level(s) increased oractin level(s) decreased as a result of therapy. The invention alsoprovides methods for deciding on the course of a therapy in a subjectundergoing therapy. Such a course of therapy is decided on the basis ofthe level(s) of gelsolin and/or the level(s) of actin.

The gelsolin or actin measurement is typically reported in ng/μl(nanograms/microliter), μM/L (micromoles/Liter), mg/dl(milligrams/deciliter), mg/L (milligrams/Liter) or μg/ml(microgram/milliliter).

The amount of a treatment may be varied for example by increasing ordecreasing the amount of gelsolin or pharmacological agent or atherapeutic composition, by changing the therapeutic compositionadministered, by changing the route of administration, by changing thedosage timing and so on. The effective amount will vary with thecondition being treated, the age and physical condition of the subjectbeing treated, the severity of the condition, the duration of thetreatment, the nature of the concurrent therapy (if any), the specificroute of administration, and like factors are within the knowledge andexpertise of the health practitioner. For example, an effective amountcan depend upon the duration the subject has had the renal failure.

An effective amount is a dosage of the therapeutic agent sufficient toprovide a medically desirable result. An effective amount may also, forexample, depend upon the degree to which a subject has abnormallydecreased levels of gelsolin and/or abnormally elevated levels of actin.It should be understood that the therapeutic agents of the invention areused, for example, to treat or prevent complications (e.g., infectiousor cardiac) in a renal failure subject. Thus, for example, an effectiveamount is that amount which can lower the risk of, slow or perhapsprevent altogether the development of an infection or a cardiaccomplication in a renal failure subject.

The factors involved in determining an effective amount are well knownto those of ordinary skill in the art and can be addressed with no morethan routine experimentation. In some embodiments, a maximum dose of thepharmacological agents of the invention (alone or in combination withother therapeutic agents) be used, that is, the highest safe doseaccording to sound medical judgment. It will be understood by those ofordinary skill in the art however, that a subject may insist upon alower dose or tolerable dose for medical reasons, psychological reasonsor for virtually any other reason(s).

The therapeutically effective amount of a pharmacological agent of theinvention is that amount effective to increase the level of gelsolinand/or decrease the level of actin or to treat or prevent an infectionor a cardiac complication in a renal failure subject. For example, thedesired response may be inhibiting the progression of an infection or acardiac complication. This may involve only slowing the progression ofthe infection or the cardiac complication temporarily, although morepreferably, it involves halting the progression of the infection or thecardiac complication. This can be monitored by routine diagnosticmethods known to those of ordinary skill in the art. The desiredresponse to treatment may be an increase in the level of plasma gelsolinor a decrease in the plasma level of actin.

The pharmacological agents used in the methods of the invention arepreferably sterile and contain an effective amount of gelsolin forproducing the desired response in a unit of weight or volume suitablefor administration to a subject. The doses of pharmacological agentsadministered to a subject can be chosen in accordance with differentparameters, in particular in accordance with the mode of administrationused and the state of the subject. Other factors include the desiredperiod of treatment. In the event that a response in a subject isinsufficient at the initial doses applied, higher doses (or effectivelyhigher doses by a different, more localized delivery route) may beemployed to the extent that patient tolerance permits. The dosage of apharmacological agent may be adjusted by the individual physician orveterinarian, particularly in the event of any complication. Atherapeutically effective amount typically varies from 0.01 mg/kg toabout 1000 mg/kg, preferably from about 0.1 mg/kg to about 500 mg/kg,and most preferably from about 0.2 mg/kg to about 250 mg/kg, in one ormore dose administrations daily, for one or more days.

Gelsolin and optionally other therapeutics may be administered per se orin the form of a pharmaceutically acceptable salt.

Various modes of administration are known to those of ordinary skill inthe art which effectively deliver the pharmacological agents of theinvention to a desired tissue, cell, or bodily fluid. The administrationmethods are discussed elsewhere in the application. The invention is notlimited by the particular modes of administration disclosed herein.Standard references in the art (e.g., Remington's PharmaceuticalSciences, 20th Edition, Lippincott, Williams and Wilkins, Baltimore Md.,2001) provide modes of administration and formulations for delivery ofvarious pharmaceutical preparations and formulations in pharmaceuticalcarriers. Other protocols which are useful for the administration ofpharmacological agents of the invention will be known to one of ordinaryskill in the art, in which the dose amount, schedule of administration,sites of administration, mode of administration and the like vary fromthose presented herein.

When administered, the pharmaceutical preparations of the invention areapplied in pharmaceutically-acceptable amounts and inpharmaceutically-acceptable compositions. The term “pharmaceuticallyacceptable” means a non-toxic material that does not interfere with theeffectiveness of the biological activity of the active ingredients. Suchpreparations may routinely contain salts, buffering agents,preservatives, compatible carriers, and optionally other therapeuticagents. When used in medicine, the salts should be pharmaceuticallyacceptable, but non-pharmaceutically acceptable salts may convenientlybe used to prepare pharmaceutically-acceptable salts thereof and are notexcluded from the scope of the invention. Such pharmacologically andpharmaceutically-acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic,succinic, and the like. Also, pharmaceutically-acceptable salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts.

A pharmacological agent or composition may be combined, if desired, witha pharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier” as used herein means one or morecompatible solid or liquid fillers, diluents or encapsulating substanceswhich are suitable for administration into a human. The term “carrier”denotes an organic or inorganic ingredient, natural or synthetic, withwhich the active ingredient is combined to facilitate the application.The components of the pharmaceutical compositions also are capable ofbeing co-mingled with the pharmacological agents of the invention, andwith each other, in a manner such that there is no interaction whichwould substantially impair the desired pharmaceutical efficacy.

The pharmaceutical compositions may contain suitable buffering agents,as described above, including: acetate, phosphate, citrate, glycine,borate, carbonate, bicarbonate, hydroxide (and other bases) andpharmaceutically acceptable salts of the foregoing compounds. Thepharmaceutical compositions also may contain, optionally, suitablepreservatives, such as: benzalkonium chloride, chlorobutanol, parabensand thimerosal.

The pharmaceutical compositions may conveniently be presented in unitdosage form and may be prepared by any of the methods well known in theart of pharmacy. All methods include the step of bringing the activeagent into association with a carrier, which constitutes one or moreaccessory ingredients. In general, the compositions are prepared byuniformly and intimately bringing the active compound into associationwith a liquid carrier, a finely divided solid carrier, or both, andthen, if necessary, shaping the product.

The compounds, when it is desirable to deliver them systemically, may beformulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active compounds may be in powder form forconstitution with a suitable vehicle (e.g., saline, buffer, or sterilepyrogen-free water) before use.

Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, pills, lozenges, eachcontaining a predetermined amount of the active compound (e.g.,gelsolin). Other compositions include suspensions in aqueous liquids ornon-aqueous liquids such as a syrup, elixir, an emulsion, or a gel.

Pharmaceutical preparations for oral use can be obtained as solidexcipient, optionally grinding a resulting mixture, and processing themixture of granules, after adding suitable auxiliaries, if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, sorbitolor cellulose preparations such as, for example, maize starch, wheatstarch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose,and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents maybe added, such as the cross-linked polyvinyl pyrrolidone, agar, oralginic acid or a salt thereof such as sodium alginate. Optionally theoral formulations may also be formulated in saline or buffers, i.e. EDTAfor neutralizing internal acid conditions or may be administered withoutany carriers.

Also specifically contemplated are oral dosage forms of the abovecomponent or components. The component or components may be chemicallymodified so that oral delivery of the derivative is efficacious.Generally, the chemical modification contemplated is the attachment ofat least one moiety to the component molecule itself, where said moietypermits (a) inhibition of proteolysis; and (b) uptake into the bloodstream from the stomach or intestine. Also desired is the increase inoverall stability of the component or components and increase incirculation time in the body. Examples of such moieties include:polyethylene glycol, copolymers of ethylene glycol and propylene glycol,carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone and polyproline. Abuchowski and Davis, 1981 “SolublePolymer-Enzyme Adducts” In: Enzymes as Drugs, Hocenberg and Roberts,eds., Wiley-Interscience, New York, N.Y., pp. 367-383; Newmark, et al.,1982 J. Appl. Biochem. 4:185-189. Other polymers that could be used arepoly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred forpharmaceutical usage, as indicated above, are polyethylene glycolmoieties.

For the component (or derivative) the location of release may be thestomach, the small intestine (the duodenum, the jejunum, or the ileum),or the large intestine. One skilled in the art has availableformulations which will not dissolve in the stomach, yet will releasethe material in the duodenum or elsewhere in the intestine. Preferably,the release will avoid the deleterious effects of the stomachenvironment, either by protection of gelsolin or by release of thebiologically active material beyond the stomach environment, such as inthe intestine.

To ensure full gastric resistance a coating impermeable to at least pH5.0 is essential. Examples of the more common inert ingredients that areused as enteric coatings are cellulose acetate trimellitate (CAT),hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50 HPMCP 55polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, celluloseacetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. Thesecoatings may be used as mixed films.

A coating or mixture of coatings can also be used on tablets, which arenot intended for protection against the stomach. This can include sugarcoatings, or coatings which make the tablet easier to swallow. Capsulesmay consist of a hard shell (such as gelatin) for delivery of drytherapeutic i.e. powder; for liquid forms, a soft gelatin shell may beused. The shell material of cachets could be thick starch or otheredible paper. For pills, lozenges, molded tablets or tablet triturates,moist massing techniques can be used.

The therapeutic can be included in the formulation as finemulti-particulates in the form of granules or pellets of particle sizeabout 1 mm. The formulation of the material for capsule administrationcould also be as a powder, lightly compressed plugs or even as tablets.The therapeutic could be prepared by compression.

Colorants and flavoring agents may all be included. For example,gelsolin may be formulated (such as by liposome or microsphereencapsulation) and then further contained within an edible product, suchas a refrigerated beverage containing colorants and flavoring agents.

One may dilute or increase the volume of the therapeutic with an inertmaterial. These diluents could include carbohydrates, especiallymannitol, lactose, anhydrous lactose, cellulose, sucrose, modifieddextrans and starch. Certain inorganic salts may be also be used asfillers including calcium triphosphate, magnesium carbonate and sodiumchloride. Some commercially available diluents are Fast-Flo, Emdex,STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the formulation of the therapeutic intoa solid dosage form. Materials used as disintegrants include but are notlimited to starch, including the commercial disintegrant based onstarch, Explotab. Sodium starch glycolate, Amberlite, sodiumcarboxymethylcellulose, ultramylopectin, sodium alginate, gelatin,orange peel, acid carboxymethyl cellulose, natural sponge and bentonitemay all be used. Another form of the disintegrants are the insolublecationic exchange resins. Powdered gums may be used as disintegrants andas binders and these can include powdered gums such as agar, Karaya ortragacanth. Alginic acid and its sodium salt are also useful asdisintegrants.

Binders may be used to hold the therapeutic agent together to form ahard tablet and include materials from natural products such as acacia,tragacanth, starch and gelatin. Others include methyl cellulose (MC),ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both beused in alcoholic solutions to granulate the therapeutic.

An anti-frictional agent may be included in the formulation of thetherapeutic to prevent sticking during the formulation process.Lubricants may be used as a layer between the therapeutic and the diewall, and these can include but are not limited to; stearic acidincluding its magnesium and calcium salts, polytetrafluoroethylene(PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricantsmay also be used such as sodium lauryl sulfate, magnesium laurylsulfate, polyethylene glycol of various molecular weights, Carbowax 4000and 6000.

Glidants that might improve the flow properties of the drug duringformulation and to aid rearrangement during compression might be added.The glidants may include starch, talc, pyrogenic silica and hydratedsilicoaluminate.

To aid dissolution of the therapeutic into the aqueous environment asurfactant might be added as a wetting agent. Surfactants may includeanionic detergents such as sodium lauryl sulfate, dioctyl sodiumsulfosuccinate and dioctyl sodium sulfonate. Cationic detergents mightbe used and could include benzalkonium chloride or benzethomiumchloride. The list of potential non-ionic detergents that could beincluded in the formulation as surfactants are lauromacrogol 400,polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and60 glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fattyacid ester, methyl cellulose and carboxymethyl cellulose. Thesesurfactants could be present in the formulation of gelsolin either aloneor as a mixture in different ratios.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added.

Microspheres formulated for oral administration may also be used. Suchmicrospheres have been well defined in the art. All formulations fororal administration should be in dosages suitable for suchadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention may be conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

Also contemplated herein is pulmonary delivery of gelsolin. Gelsolin isdelivered to the lungs of a mammal while inhaling and traverses acrossthe lung epithelial lining to the blood stream. Other reports of inhaledmolecules include Adjei et al., 1990, Pharmaceutical

Research, 7:565-569; Adjei et al., 1990 International Journal ofPharmaceutics, 63:135-144 (leuprolide acetate); Braquet et al., 1989Journal of Cardiovascular Pharmacology, 13(suppl. 5):143-146(endothelin-1); Hubbard et al., 1989 Annals of Internal Medicine, Vol.III, pp. 206-212 (al-antitrypsin); Smith et al., 1989 J. Clin. Invest.84:1145-1146 (a-1-proteinase); Oswein et al., 1990 “Aerosolization ofProteins”, Proceedings of Symposium on Respiratory Drug Delivery II,Keystone, Colo., March, (recombinant human growth hormone); Debs et al.,1988 J. Immunol. 140:3482-3488 (interferon-y and tumor necrosis factoralpha) and Platz et al., U.S. Pat, No. 5,284,656 (granulocyte colonystimulating factor). A method and composition for pulmonary delivery ofdrugs for systemic effect is described in U.S. Pat. No. 5,451,569 issuedSep. 19, 1995 to Wong et al.

Contemplated for use in the practice of this invention are a wide rangeof mechanical devices designed for pulmonary delivery of therapeuticproducts, including but not limited to nebulizers, metered doseinhalers, and powder inhalers, all of which are familiar to thoseskilled in the art.

Some specific examples of commercially available devices suitable forthe practice of this invention are the Ultravent nebulizer, manufacturedby Mallinckrodt, Inc., St. Louis, Missouri; the Acorn II nebulizer,manufactured by Marquest Medical Products, Englewood, Colorado; theVentolin metered dose inhaler, manufactured by Glaxo Inc., ResearchTriangle Park, N.C.; and the Spinhaler powder inhaler, manufactured byFisons Corp., Bedford, Mass.

All such devices require the use of formulations suitable for thedispensing of gelsolin. Typically, each formulation is specific to thetype of device employed and may involve the use of an appropriatepropellant material, in addition to the usual diluents, adjuvants and/orcarriers useful in therapy. Also, the use of liposomes, microcapsules ormicrospheres, inclusion complexes, or other types of carriers iscontemplated. Chemically modified gelsolin may also be prepared indifferent formulations depending on the type of chemical modification orthe type of device employed.

Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise gelsolin dissolved in water at aconcentration of about 0.1 to 25 mg of biologically active gelsolin permL of solution. The formulation may also include a buffer and a simplesugar (e.g., for gelsolin stabilization and regulation of osmoticpressure). The nebulizer formulation may also contain a surfactant, toreduce or prevent surface induced aggregation of the gelsolin caused byatomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device will generallycomprise a finely divided powder containing the gelsolin suspended in apropellant with the aid of a surfactant. The propellant may be anyconventional material employed for this purpose, such as achlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or ahydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, orcombinations thereof. Suitable surfactants include sorbitan trioleateand soya lecithin. Oleic acid may also be useful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise afinely divided dry powder containing gelsolin and may also include abulking agent, such as lactose, sorbitol, sucrose, or mannitol inamounts which facilitate dispersal of the powder from the device, e.g.,50 to 90% by weight of the formulation. The gelsolin should mostadvantageously be prepared in particulate form with an average particlesize of less than 10 mm (or microns), most preferably 0.5 to 5 mm, formost effective delivery to the distal lung.

Nasal (or intranasal) delivery of a pharmaceutical composition of thepresent invention is also contemplated. Nasal delivery allows thepassage of a pharmaceutical composition of the present invention to theblood stream directly after administering the therapeutic product to thenose, without the necessity for deposition of the product in the lung.Formulations for nasal delivery include those with dextran orcyclodextran.

For nasal administration, a useful device is a small, hard bottle towhich a metered dose sprayer is attached. In one embodiment, the metereddose is delivered by drawing the pharmaceutical composition of thepresent invention solution into a chamber of defined volume, whichchamber has an aperture dimensioned to aerosolize and aerosolformulation by forming a spray when a liquid in the chamber iscompressed. The chamber is compressed to administer the pharmaceuticalcomposition of the present invention. In a specific embodiment, thechamber is a piston arrangement. Such devices are commerciallyavailable.

Alternatively, a plastic squeeze bottle with an aperture or openingdimensioned to aerosolize an aerosol formulation by forming a spray whensqueezed is used. The opening is usually found in the top of the bottle,and the top is generally tapered to partially fit in the nasal passagesfor efficient administration of the aerosol formulation. Preferably, thenasal inhaler will provide a metered amount of the aerosol formulation,for administration of a measured dose of the drug.

The compounds may also be formulated in rectal or vaginal compositionssuch as suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be formulated with suitable polymeric or hydrophobic materials (forexample as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

Suitable liquid or solid pharmaceutical preparation forms are, forexample, aqueous or saline solutions for inhalation, microencapsulated,encochleated, coated onto microscopic gold particles, contained inliposomes, nebulized, aerosols, pellets for implantation into the skin,or dried onto a sharp object to be scratched into the skin. Thepharmaceutical compositions also include granules, powders, tablets,coated tablets, (micro)capsules, suppositories, syrups, emulsions,suspensions, creams, drops or preparations with protracted release ofactive compounds, in whose preparation excipients and additives and/orauxiliaries such as disintegrants, binders, coating agents, swellingagents, lubricants, flavorings, sweeteners or solubilizers arecustomarily used as described above. The pharmaceutical compositions aresuitable for use in a variety of drug delivery systems. For a briefreview of methods for drug delivery, see Langer, Science 249:1527-1533,1990 which is incorporated herein by reference.

The therapeutic agent(s), including specifically but not limited togelsolin, may be provided in particles. Particles as used herein meansnano or micro particles (or in some instances larger) which can consistin whole or in part of gelsolin or the other therapeutic agent(s) asdescribed herein. The particles may contain the therapeutic agent(s) ina core surrounded by a coating, including, but not limited to, anenteric coating. The therapeutic agent(s) also may be dispersedthroughout the particles. The therapeutic agent(s) also may be adsorbedinto the particles. The particles may be of any order release kinetics,including zero order release, first order release, second order release,delayed release, sustained release, immediate release, and anycombination thereof, etc. The particle may include, in addition to thetherapeutic agent(s), any of those materials routinely used in the artof pharmacy and medicine, including, but not limited to, erodible,nonerodible, biodegradable, or nonbiodegradable material or combinationsthereof. The particles may be microcapsules which contain the gelsolinin a solution or in a semi-solid state. The particles may be ofvirtually any shape.

Both non-biodegradable and biodegradable polymeric materials can be usedin the manufacture of particles for delivering the therapeutic agent(s).Such polymers may be natural or synthetic polymers. The polymer isselected based on the period of time over which release is desired.Bioadhesive polymers of particular interest include bioerodiblehydrogels described by H. S. Sawhney, C. P. Pathak and J. A. Hubell inMacromolecules (1993) 26:581-587 the teachings of which are incorporatedherein. These include polyhyaluronic acids, casein, gelatin, glutin,polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methylmethacrylates), poly(ethyl methacrylates), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), and poly(octadecyl acrylate).

The therapeutic agent(s) may be contained in controlled release systems.The term “controlled release” is intended to refer to anydrug-containing formulation in which the manner and profile of drugrelease from the formulation are controlled. This refers to immediate aswell as non-immediate release formulations, with non-immediate releaseformulations including but not limited to sustained release and delayedrelease formulations. The term “sustained release” (also referred to as“extended release”) is used in its conventional sense to refer to a drugformulation that provides for gradual release of a drug over an extendedperiod of time, and that preferably, although not necessarily, resultsin substantially constant blood levels of a drug over an extended timeperiod. The term “delayed release” is used in its conventional sense torefer to a drug formulation in which there is a time delay betweenadministration of the formulation and the release of the drug therefrom. “Delayed release” may or may not involve gradual release of drugover an extended period of time, and thus may or may not be “sustainedrelease.”

Use of a long-term sustained release implant may be particularlysuitable for treatment of chronic conditions. “Long-term” release, asused herein, means that the implant is constructed and arranged todeliver therapeutic levels of the active ingredient for at least 7 days,and preferably 30-60 days. Long-term sustained release implants arewell-known to those of ordinary skill in the art and include some of therelease systems described above.

For topical administration to the eye, nasal membranes, mucous membranesor to the skin, the gelsolin may be formulated as ointments, creams orlotions, or as a transdermal patch or intraocular insert oriontophoresis. For example, ointments and creams can be formulated withan aqueous or oily base alone or together with suitable thickeningand/or gelling agents. Lotions can be formulated with an aqueous or oilybase and, typically, further include one or more emulsifying agents,stabilizing agents, dispersing agents, suspending agents, thickeningagents, or coloring agents. (See, e.g., U.S. Pat. No. 5,563,153,entitled “Sterile Topical Anesthetic Gel”, issued to Mueller, D., etal., for a description of a pharmaceutically acceptable gel-basedtopical carrier.)

In general, the gelsolin or the actin-binding molecule is present in atopical formulation in an amount ranging from about 0.01% to about 30.0%by weight, based upon the total weight of the composition. Preferably,the gelsolin is present in an amount ranging from about 0.5 to about 30%by weight and, most preferably, the gelsolin is present in an amountranging from about 0.5 to about 10% by weight. In one embodiment, thecompositions of the invention comprise a gel mixture to maximize contactwith the surface of the localized pain and minimize the volume anddosage necessary to alleviate the localized pain. GELFOAM ® (amethylcellulose-based gel manufactured by Upjohn Corporation) is apreferred pharmaceutically acceptable topical carrier. Otherpharmaceutically acceptable carriers include iontophoresis fortransdermal drug delivery.

The invention also contemplates the use of kits. In some aspects of theinvention, the kit can include a pharmaceutical preparation vial, apharmaceutical preparation diluent vial, and gelsolin. The vialcontaining the diluent for the pharmaceutical preparation is optional.The diluent vial contains a diluent such as physiological saline fordiluting what could be a concentrated solution or lyophilized powder ofgelsolin. The instructions can include instructions for mixing aparticular amount of the diluent with a particular amount of theconcentrated pharmaceutical preparation, whereby a final formulation forinjection or infusion is prepared. The instructions may includeinstructions for treating a subject with an effective amount ofgelsolin. It also will be understood that the containers containing thepreparations, whether the container is a bottle, a vial with a septum,an ampoule with a septum, an infusion bag, and the like, can containindicia such as conventional markings which change color when thepreparation has been autoclaved or otherwise sterilized.

The present invention is further illustrated by the following Example,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

EXAMPLE

Overview:

Accelerated Mortality on Renal Replacement (ArMORR) is a nationallyrepresentative prospective cohort study of patients that initiatedchronic hemodialysis at U.S. dialysis centers operated by FreseniusMedical Care, North America (FMC, Lexington, Mass.). Informationcollected prospectively included patient demographics, comorbidities atthe initiation of hemodialysis, laboratory tests (performed by SpectraEast, Rockland, N.J.), intravenous therapies, and clinical outcomes.Data were entered into a central database by physicians and nurses atthe point of care, with rigorous quality assurance/quality controlauditing mandated by FMC^(22, 23). This study was approved by theInstitutional Review Board of the Massachusetts General Hospital.

Study Population:

Between Jul. 1, 2004 and Jun. 30, 2005, 10,044 incident hemodialysispatients representing 1056 U.S. dialysis units were prospectivelyenrolled into ArMORR. All incident hemodialysis patients who initiatedtherapy at a US-based Fresenius unit were eligible for inclusion in theArMORR cohort. A random sample of 150 patients with an availablebaseline (collected within 14 days of initiating chronic hemodialysis)blood sample for plasma gelsolin and actin, and serum high sensitivity Creactive protein were included in the study. Of these 150 subjects, 41(27%) died within 365 days of initiating dialysis and 109 survived forat least 365 days. To efficiently study the effects of pGSN levels onsurvival, we also performed a nested case-control study defining casesas ESRD subjects who died within 365 days of initiating dialysis andcontrols as those who survived for at least 365 days. To increase power,we added the next 75 consecutive ArMORR participants who died within 365days of initiating dialysis (n=116 total cases) to the original sampleto create a case-control sample of ˜1:1 ratio with a total of 225subjects. We aimed to include a similar number of CVD deaths andinfectious deaths (defined below). Subsequently, 2 patients were foundnot to have sufficient blood sample, hence they were excluded, leaving atotal of 223 subjects to study. With a case-control sample of 223 and a˜1:1 ratio, we had >80% power to detect an odds ratio of at least 2among patients with pGSN deficiency (e.g., lowest category if examinedin tertiles) compared to those with higher levels.

Ascertainment of Exposures and Outcomes:

The primary exposure was baseline pGSN levels, and the primary outcomewas overall one-year mortality. pGSN was examined as a continuous andbinomial (based on the median levels in the random) variable, and weexamined pGSN in tertile analyses. In addition to overall mortality, wealso defined outcomes with cardiovascular (e.g., died of diseases of thecirculatory system, ICD-9 390-459.9; hypertensive diseases, 401-405;ischemic heart disease, 410-414; acute myocardial infarction, 410; andcerebrovascular disease, 430-438) and infectious causes of mortality(e.g., bacterial, fungal, and viral pneumonias, ICD-9 480.0-487.8;empyema, 510.0; lung abscess, 513.0; sepsis, severe sepsis, and septicshock, 038, 995-996, 785) within one year of initiating chronic HD.Death was confirmed by discharge diagnosis reports from the individualdialysis centers.

The primary covariate of interest was plasma actin levels that weresemi-quantitated (see below). Other covariates included age, race, sex,body mass index, assigned cause of renal failure (e.g., diabetes,hypertension, glomerulonephritis, polycystic kidney disease or other),blood pressure, body mass index, vascular access at initiation(arteriovenous fistula, graft or veno-venous catheter), and dialysisdose (Kt/V) as we have done in prior analyses^(22, 23). Baseline bloodlevels of albumin, creatinine, calcium, phosphorus, and platelet andwhite blood cell count were analyzed. Serum level of high sensitivityCRP (hsCRP) at baseline using standard techniques (N Latex CRP assay,Dade Behring) was also measured.

Plasma Gelsolin (pGSN):

pGSN was measured by its ability to stimulate actin nucleation asdescribed previously²⁴. This functional assay is highly reproducible anddetects total levels of pGSN irrespective of whether it is complexed toactin or other pGSN ligands. In brief, baseline plasma was diluted 1:5fold in 0.1 M KCl, 0.2 mM MgCl₂, 1 mM EGTA, 0.5 mM ATP, 0.5 mMβ-mercaptoethanol, and 10 mM Tris-HCl buffer, pH 7.4 (Buffer B). Of thediluted plasma sample, 5 μl was added to 280μl Buffer B supplementedwith 1.5 mM CaCl₂ and 0.4 μM Phallacidin in 6×50 mm borosilicate culturetubes. The actin polymerization reaction was initiated by adding 15 μl20 μM pyrene actin²⁵ in 0.5 mM ATP, 5 mM β-mercaptoethanol, 0.2 mMCaCl₂, 0.2 mM Tris-HCl buffer, pH 7.4 (Buffer A). Polymerization wasmonitored for 200 seconds in a spectrofluorimeter at excitation andemission wavelengths of 366 and 386 nm respectively. pGSN concentrationswere estimated from a standard curve using purified recombinant humanpGSN synthesized in E. coli. All measurements were performed with thelaboratory technician blinded to the outcomes.

Detecting Circulating Actin:

Plasma was diluted 1:10 fold in phosphate buffered saline (PBS) and thenanalyzed by E-PAGE 48 8% gel system as per manufacturer's instructions(Invitrogen, Carlsbad, Calif.). Each sample was heated at 70° C. for 10minutes in β-mercaptoethanol-containing sample buffer prior to loadingonto an E-PAGE 48 gel, then transferred to nitrocellulose membranes.After blocking the membrane in 5% non-fat dry milk in Tris-bufferedsaline (TBS) with 0.05% Tween 20 primary anti-β actin antibodies (AC-15,Sigma, St. Louis, Mo.) were added at 1:2000 dilution and incubated atroom temperature for 1 hr. Bound primary antibodies were probed withHRP-linked anti-rabbit IgG's (Santa Cruz Biotechnology, Santa Cruz,Calif.) at 1:2000 dilution. Chemiluminescence of HRP was developed withSuper Signal West Pico Kit (Pierce, Rockford, Ill). The presence ofactin was defined as the appearance on the blots of discrete bandsco-migrating with purified rabbit skeletal muscle actin (Cytoskeleton,Denver, Colo.). The specificity of the actin on the Western blots wasconfirmed by subjecting 10 randomly selected samples to massspectrometry (Beth Israel Deaconess Medical Center Mass SpectrometryCore Facility). All measurements were performed with the laboratorytechnician blinded to the outcomes.

Statistical Analyses:

We used two-sample t tests and Fisher's exact to compare demographic andlaboratory characteristics and pGSN and levels and the presence of actinat the initiation of dialysis among the patients who died and those thatdid not. To examine whether routine laboratory tests were associatedwith pGSN levels, we used Spearman correlation coefficients. We usedlinear regression models to examine independent relationships betweenpGSN and other covariates. Univariate analysis of survival was performedon the initial random sampling of 150 subjects using Kaplan-Meier curveswith log-rank tests after dividing baseline values of pGSN into binaryor tertile values. The total number of subjects censored for recovery ofrenal function, kidney transplantation, or lost to follow up becausethey transferred their care to a non-FMC center was less than 8%.

Multivariate logistic regression models were used to examine theindependent association between baseline pGSN and all-cause,cardiovascular, and infectious causes of one-year mortality. We includedcovariates in the multivariate models that have been associated withmortality on dialysis in previous studies^(22, 23) and those that weresignificantly different among cases and controls in the current study.We also adjusted all models for C-reactive protein levels given theirrelationship with vascular disease and mortality among hemodialysispatients⁵. Data points on individual covariates were missing in <5% ofsubjects; for the multivariate analyses, these covariates were treatedas categorical variables with an additional category for missing values.Otherwise, continuous variables were analyzed on a continuous scale. Weexamined the relationship between pGSN and outcomes stratified by plasmalevels of actin given the biological relationship of these two measures.Finally, first order interactions were examined between pGSN andcovariates (pGSN×covariate) in univariate and multivariate models, andwhen significant (p≤0.1) interaction was detected, stratified modelswere presented. Finally, Analyses were performed using SAS 9.1 (Cary,N.C.) and two-sided p-values<0.05 were considered statisticallysignificant.

Results:

Baseline Characteristics: The initial sample of 150 ESRD subjectsrepresented 148 separate dialysis centers across the U.S. The baselinecharacteristics of these subjects are presented in Table 1 and resemblebaseline characteristics of larger populations of ESRD subjects at theinitiation of chronic hemodialysis²⁶. The distribution of baseline pGSNlevels is shown in FIG. 1. Mean pGSN levels were 140±42 mg/L, and only 2(1%) of subjects demonstrated baseline levels at or above 250 mg/L, themean level reported in healthy volunteers (dashed line in FIG. 1 andTable 5)^(14, 27). Plasma gelsolin levels correlated inversely with age(r=−0.18 p<0.01) and baseline measures of muscle mass and nutrition,such as serum creatinine (r=0.26 p<0.01) and albumin levels (r=0.34p<0.01). The correlation between pGSN and body mass index was 0.02(p>0.05). When baseline hs-CRP levels were examined in tertiles, thosewith the lowest levels of hs-CRP demonstrated the highest levels ofpGSN: tertile 1 hs-CRP<12 mg/L, pGSN 145±39 mg/L; tertiles 2 & 3hs-CRP≥12 mg/L, pGSN 131±53 mg/L, P=0.048). Linear regression analysesconfirmed that among the continuous variables in Table 1 that met a pvalue threshold of 0.1, only scrum albumin was independently correlatedwith pGSN levels (p<0.01).

Plasma pGSN and One Year Survival: The median pGSN level among the 150subjects was 141 mg/L (IQR 116-161 mg/L). Kaplan-Meier analyses of1-year survival according to binary pGSN levels (<or≥141 mg/L)demonstrated a significant survival difference according to baselinepGSN levels (FIG. 2—top panel). Similarly, dividing pGSN into tertilesrevealed a graded relationship with baseline pGSN levels and one-yearmortality (FIG. 2—bottom panel). The median day of death among thosethat died within one-year was 188 days (IQR 89-297 days).

The case-control sample of 223 patients was subsequently utilized toexamine one-year survival. Baseline characteristics according toone-year outcomes are presented in Table 2. Those who died within oneyear were slightly older, were more likely to have an intravenouscatheter as their initial vascular access (compared with arterio-venousfistula or graft), and had lower serum albumin and higher white bloodcell counts at baseline. These baseline differences have been reportedin previous studies of hemodialysis mortality²⁶. Mean pGSN levels weresignificantly lower in patients who died (117±38 mg/L) compared tosurvivors (147±42 mg/L, p<0.001). Baseline pGSN levels did not differbetween cardiovascular (n=59, 116±41 mg/L) and infectious (n=55, 117±34mg/L, p=0.91) deaths.

Multivariable Analysis of Mortality: We next examined the relationshipof pGSN levels and 1-year mortality after adjusting for importantcovariates and potential confounders (Table 3). For every 10 mg/Lreduction in baseline pGSN, the risk for subsequent mortality wasincreased by 15% (95% CI, 7-23%). The risk among those with the lowestbaseline levels (tertile 1, <130 mg/L) demonstrated the highest risk forone-year all cause and infectious causes of mortality. Both findingswere significant and demonstrated a strong linear trend. The results forcardiovascular causes of death were less significant. In these analyses,hs-CRP did not significantly associate with one-year mortality. Inaddition, serum creatinine, which was significant on univariateanalysis, was no longer significant once the model was adjusted forpGSN.

Serum albumin, a measure of nutrition and muscle mass, has been stronglyassociated with ESRD mortality²⁸. We then examined the effect of serumalbumin on the multivariable models and noted that while the pointestimates for each tertile of pGSN were modestly larger without serumalbumin, the level and direction of significance did not change byadding serum albumin. Alternatively, including or excluding pGSN gavethe following results with serum albumin: excluding pGSN, tertile 1(serum albumin<3.2 mg/dl), OR 3.0, 95% CI 1.1-6.4; tertile 2 (3.2-3.6mg/dl), OR 1.1, 0.5-2.4; tertile 3 (>3.6 mg/dl), OR 1.0 (ref); includingpGSN, tertile 1 OR 2.0, 95% CI 0.8-4.9; tertile 2 OR 1.0, 0.5-2.4;tertile 3 OR 1.0 (ref). When serum albumin was modeled as a continuousvariable, it remained significant even after adjustment for pGSN(excluding pGSN, OR 0.32 for each 1 mg/dl increase of serum albumin, 95%CI 0.15-0.67; including pGSN, OR 0.39, 95% CI 0.18-0.83). Therefore,addition of pGSN to the models attenuated but did not extinguishassociation between serum albumin and mortality.

Circulating Actin, pGSN, and Mortality: Western blotting was used todetect plasma actin. Although actin polypeptides were clearly visible asdiscrete bands on the blots, and these bands were verified as authenticactin by mass spectrometry, the presence of non-specific backgroundstaining due to high plasma protein concentrations and the relativelylow affinity of anti-actin antibodies precluded detailed quantificationof actin protein in the samples. Sixty-nine percent of patients hadcirculating actin at baseline, and diabetic renal failure patients weremore likely to have circulating actin (85%) than patients with othercauses of renal failure (59%, P<0.001). Compared to those with no actin,pGSN levels were lower in patients with actin (141±36 mg/L vs. 127±45mg/L, respectively, P=0.02) (FIG. 3) which was consistent with previousresults in sepsis samples¹⁰.

We therefore examined the relationship of baseline plasma actin(presence vs. absence) and 1-year mortality. In univariate analysis, thepresence of actin conferred a 3.5 fold (95% CI 1.9-6.4) increase in riskfor death at one year. This relationship persisted on multivariateanalyses (OR 4.6, 95% CI 2.0-10.5). The presence of diabetic renalfailure, which significantly associated with early mortality onunivariate analyses (OR 1.8, 95% CI 1.1-3.0), became non-significantafter adjusting for circulating actin (OR 1.3, 95% CI 0.7-2.3). Giventhat pGSN binds actin released by tissue damage and may abrogateactin-induced injury,^(14, 20, 27, 42) we hypothesized that low pGSN andelevated actin would increase risk of adverse outcomes.

We next examined the risk for one-year mortality according to pGSNlevels and presence or absence of actin (FIG. 4). In these analyses,pGSN was divided into a binomial variable as above. These resultssuggested that the combined parameters of low pGSN and detectable actinwere potentially associated synergistically rather than additively torisk of death.

Veno-Venous Catheter and Mortality: We sought additional effectmodifications by including interaction terms (e.g., pGSN×covariate) inmultivariable models with covariates of interest. The only additionalinteraction suggested was vascular access type (P=0.04). Althoughveno-venous catheter vascular access associates with an increased riskfor early mortality²⁹, deciphering those most susceptible to death hasbeen challenging. Patients initiating hemodialysis with a catheter(129±49 mg/L) or with an arterio-venous fistula or graft (136±32 mg/L,P=0.24) did not differ at baseline by pGSN levels, nor by the frequencyof circulating actin (71% vs. 68%, respectively, P=0.67). Nevertheless,a veno-venous catheter appeared to influence one-year mortality risk(Table 4). Amongst patients with a veno-venous catheter, those with lowpGSN and detectable circulating actin had a marked increase in overallmortality compared to those with high pGSN and no detectable actin (OR25.9, 95% CI 4.3-157.0).

pGSN, Circulating Actin, and Chronic Kidney Disease: pGSN levelscorrelated directly with estimated glomerular filtration rate (r=0.39,P=0.003) in subjects with chronic kidney disease not on dialysis (FIG.5). Males (153±43 mg/L) tended to have higher levels of pGSN comparedwith females (136±52 mg/L, p=0.09). Levels in late stages of kidneydisease (e.g., stages 3 and 4) were comparable to those found at theinitiation of chronic hemodialysis. However, these levels weresignificantly lower than in samples obtained from stages 1 and 2(P=0.002) (FIG. 6). The frequency of circulating actin was 11% in thispre-dialysis cohort, in contrast to 69% in the dialysis cohort(P<0.001).

Discussion:

Patients initiating hemodialysis have pGSN levels reduced to an average30-50% lower than found in healthy controls. pGSN declines withprogressive renal disease, suggesting mechanisms upstream of chronicdialysis initiation account for pGSN reduction. Following the initiationof chronic hemodialysis, pGSN demonstrated a graded, inverserelationship with adverse outcomes—the lower the level, the higher therisk for one-year mortality.

It is believed that pGSN sequestration at sites of injury or clearancewith circulating actin may be the principal causes of decreased pGSNconcentrations following acute insults. These factors may alsocontribute to diminished pGSN in ESRD, but, in addition, impairment ofsynthesis may be important. For example, uremia is characterized byincreased activity of the ubiquitin-proteasome pathway⁴³, and recentlyincreased activity of this pathway has been linked to increaseddegradation of cGSN, the intracellular isoform of pGSN.⁴⁴ Moreover,since the molecular weight of pGSN is ˜93 kDA, pGSN is unlikely to becleared by hemodialysis. As highlighted in FIG. 7, combination ofdecreased production and increased consumption due to ongoing tissueinjury in dialysis patients are some possible etiologies of thedecreased circulating levels of pGSN in end-stage renal failuresubjects. pGSN synthesis is constitutive and does not increase likeacute phase reactants in inflammation.³⁵ Since muscle is a major sourceof pGSN, correlations with serum albumin and creatinine suggestprotein-energy wasting characteristic of ESRD may contribute to pGSNreduction.^(4, 8, 28, 36, 37, 38, 43, 45, 46) pGSN attenuates theotherwise strong relationship between serum creatinine and albumin andhemodialysis mortality,⁸ suggesting at least a partial overlap betweenthese parameters in explaining mortality.

Patients at greatest risk for death with the lowest pGSN levels werethose with detectable circulating actin. Actin has been detectable inplasma of patients with acute lung or liver injury,¹⁴ patients withsevere trauma, and even in healthy blood donors.⁴⁷ Circulating actin inover two-thirds of hemodialysis patients is consistent with wide spreadtissue injury and excess muscle protein catabolism reported in patientswith ESRD.^(43, 48, 49, 50) Most (85%) patients with diabetic renalfailure, a group with widespread endothelial cell injury and markedlyelevated mortality rates,^(1, 51, 52) had circulating actin, and it wasinteresting to find that adjustment for circulating actin eliminated therelationship between diabetes status and mortality that has beenpreviously reported.⁴¹ Circulating actin has been documented in patientswith acute respiratory distress syndrome²⁰ and in animal models ofsepsis (Table 5).¹¹ In contrast to pGSN depletion, detectablecirculating actin was far less prevalent in advanced renal disease priorto dialysis, suggesting that dialysis itself, possibly resulting fromacute hemodynamic fluxes or dialysis membrane bioincompatibilies⁵³, maycontribute to tissue damage releasing actin into the circulation.

pGSN depletion may link muscle wasting, tissue injury, inflammation anddeath due to cardiovascular events and sepsis in ESRD. pGSN depletionmay indeed characterize other chronic wasting states. pGSN avidly bindsinflammatory mediators including platelet-activating factor,lysophosphatidic acid, lipoteichoic acid, aβ peptide andlipopolysaccharide endotoxin and decreases the effects of these agonistson target cells.^(12, 30, 31, 32, 33) Loss of buffering of thesemediators due to pGSN depletion could exacerbate vascular disease andits contribution to mortality. Toxic effects of circulating actin on thevasculature might also be important.^(20, 54) Deficiency of pGSN mayalso worsen the outcome of superimposed infection. ^(10, 11, 34) LowpGSN and circulating actin conferred a markedly increased risk for earlymortality in catheter compared to graft-or fistula-managed patients.Attenuation of pGSN's ability to disrupt actin-containing biofilms maybe one mechanism low pGSN and elevated actin predispose to adverseoutcomes in catheter-instrumented patients.^(55, 56) Moreover, actinimpairs the activity of leukocyte-derived cationic anti-microbialpolypeptides known as defensins.⁴²

TABLE 1 Table 1. Baseline Characteristics N = 150 Age (Years) 64 ± 15Female (%) 45 Race (%) White 53 Black 41 Other  6 BMI (kg/m2) 28 ± 19Diabetes Mellitus (%) 20 Etiology of Renal Failure (% Diabetes) 43Vascular Access (% Catheter) 57 Systolic blood pressure (mmHg) 145 ± 20 Diastolic blood pressure (mmHg) 74 ± 13 Albumin (g/dl) 3.5 ± 0.4 Calcium(mg/dl) 8.5 ± 0.8 Phosphorus (mg/dl) 4.6 ± 1.4 Creatinine (mg/dl) 6.4 ±2.6 eKT/V 1.3 ± 0.4 Hemoglobin (g/dl) 10.0 ± 1.4  hs-C reactive protein(mg/L) 29 ± 38 White blood cell count (cells/mcl) 7.5 ± 2.6 Platelets(cells/dl) 236 ± 95 

TABLE 2 Baseline Characteristics of the Case-Control Sample* CasesControls N N = 114 N = 109 p-value Age (Years) 67 ± 13 63 ± 15 0.02Female (%) 45 45 0.99 Race (%) 0.22 White 59 47 Black 36 47 Other  5  6BMI (kg/m2) 26 ± 3  27 ± 7  0.20 Diabetes Mellitus (%) 21 20 0.86Etiology of Renal Failure 50 36 0.03 (% Diabetes) Vascular Access (%Catheter) 70 46 <0.01 Systolic blood pressure (mmHg) 140 ± 25  145 ± 20 0.02 Diastolic blood pressure (mmHg) 71 ± 14 74 ± 12 0.05 Albumin (g/dl)3.2 ± 0.6 3.5 ± 0.5 <0.01 Calcium (mg/dl) 8.3 ± 0.7 8.4 ± 0.8 0.20Phosphorus (mg/dl) 4.4 ± 1.4 4.6 ± 1.3 0.05 Creatinine (mg/dl) 5.5 ± 2.66.5 ± 2.6 0.01 eKt/V 1.3 ± 0.3 1.3 ± 0.4 0.56 Hemoglobin (g/dl) 10.1 ±1.3  10.0 ± 1.4  0.40 **hs-C reactive protein (mg/L) 20 (7-47) 13 (3-24)0.20 White blood cell count (cells/mcl) 8.7 ± 4.1 7.5 ± 2.6 0.01Platelets (cells/dl) 210 ± 84  236 ± 95  0.08 *Values are frequencies ormeans ± standard deviations. **hs-C reactive protein (hs-CRP) reportedas median values and interquartile range (IQR, 25%-75%).

TABLE 3 Multivariate risk (odds ratio) of one-year mortality accordingto tertiles of pGSN and All Cause, CVD, and Infectious causes of deathat one year. Risk for All Cause Death Odds Ratio* 95% CI P valueTertiles of pGSN Tertile 1 ≥150 mg/L 1.0 (ref) Tertile 2 130-149 mg/L2.1 0.7-6.7 0.19 Tertile 3 <130 mg/L 3.4 1.2-9.4 0.01 P for Trend = 0.01Risk for CVD Deaths Tertile 1 ≥150 mg/L 1.0 (ref) Tertile 2 130-149 mg/L1.4 0.3-5.2 0.65 Tertile 3 <130 mg/L 2.4 0.6-8.2 0.10 P for Trend = 0.05Risk for Infectious Deaths Tertile 1 ≥150 mg/L 1.0 (ref) Tertile 2130-149 mg/L 3.2  0.7-15.5 0.13 Tertile 3 <130 mg/L 5.4  1.3-22.5 0.03 Pfor trend = 0.01 *Model adjusted for baseline Age, Gender, Race, BMI,Cause of ESRD, Blood Pressure, Vascular Access, and baseline serumAlbumin, Calcium, Phosphorus, Creatinine, WBC, Platelet Count, and hsC-reactive protein.

TABLE 4 Multivariable risk (Odds Ratio) of one-year mortality accordingto Veno-Venous Catheter status at baseline, and pGSN and actinstatus.*Elevated baseline pGSN (pGSN ≥141 mg/L, +pGSN), low baselinepGSN (pGSN <141 mg/L, −pGSN); No Detectable Actin (−Actin); DetectableActin (+Actin). Cases Controls Odds Ratio* 95% CI No Veno-VenousCatheter (n = 93) +pGSN, −Actin 4 14 1.0 (ref) −pGSN, −Actin 3 8 0.30.1-7.2  +pGSN, +Actin 5 16 1.0 0.2-7.9  −pGSN, +Actin 22 21 2.40.5-12.1 Veno-Venous Catheter (n = 130) +pGSN, −Actin 4 12 1.0 (ref)−pGSN, −Actin 11 12 3.9 0.6-26.4 +pGSN, +Actin 15 14 11.1 1.8-69.5−pGSN, +Actin 50 12 25.9  4.3-157.0 *Model adjusted for baseline Age,Gender, Race, Body Mass Index, Cause of ESRD, Blood Pressure, VascularAccess, and baseline serum Albumin, Calcium, Phosphorus, Creatinine,White Blood Cell Count, Platelet Count, and high sensitivity C-reactiveprotein.

TABLE 5 Levels of plasma gelsolin (mg/L) in clinical states Mean Me-Source N (range/SD) dian Methodology Normal Dahl, et al. (1999)⁵⁸ 25 207(151-621) 200 Nephelometry DiNubile, et al. (1998)⁵⁹ 11  440 ± 150Western blot Ito, et al. (1992)⁶⁰ 43 226 ± 52 220 ELISA Smith, et al.(1987)⁶¹ 56 240 ± 50 250 ELISA, nucleation Mounzer, et al. (1999)¹⁸ 11 517 ± 134 500 Western blot Smith, et al. (1988)⁶² Healthy Gambianchildren 11 367 ± 67 Nucleation Convalescent from malaria 11  263 ± 160240 Nucleation Suhler, et al. (1997)¹⁶ 25 260 ± 20 Western blot Acutelung injury Lind, et al. (1988)⁶³† 20  89 ± 33 86 Nucleation Fulminanthepatic necrosis Suhler, et al. (1997)¹⁶ 18 100 ± 15 Western blot Acutehepatitis Ito, et al. (1992)⁶⁰† 14 80 ± 40 80 ELISA Post-hematopoieticstem call transplantation with death from interstitial pneumoniaDiNubile, et al. (1998)⁵⁹ 9 100 ± 50 Western blot Acute myocardialinfarction Suhler, et al. (1997)¹⁶ 10 180 ± 20 Western blotRhabdomyolysis Suhler, et al. (1997)¹⁶ 12 170 ± 20 Western blot Lofberg,et al. (1998)⁶⁴ 5 116 ± 22 100 RIA Bacterial pneumonia Smith, et al.(1988)⁶² 8 116 ± 89 Nucleation Lind, et al. (1988)⁶³ 6 117 ± 21 115Nucleation Sepsis Suhler, et al. (1997)¹⁶ 6 130 ± 20 Acute falciparummalaria Smith, et al. (1988)⁶² 18 126 ± 45 Nucleation Major trauma,surgery, burns Lee, et al. (2006)¹¹ Overall 31 73 70 Nucleation ICUsurvivors 28 81 (20-181) Nucleation ICU non-survivors 3 26 (25-60) Nucleation Dahl, et al. (1999)⁵⁸ 23 51 (7-967)  55 Nephelometry Mounzer,et al. (1999)¹⁸ 64 339 ± 82 290 Western blot All values are in mg/L.Unless otherwise noted, effects to detect actin were not performed.†Actin detected in plasma. ‡Actin not detected in plasma.

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EQUIVALENTS

The foregoing written specification is considered to be sufficient toenable one ordinarily skilled in the art to practice the invention. Thepresent invention is not to be limited in scope by the example(s)provided, since the example(s) are intended as mere illustrations of oneor more aspects of the invention. Other functionally equivalentembodiments are considered within the scope of the invention. Variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description. Each of the limitations of the invention canencompass various embodiments of the invention. It is, therefore,anticipated that each of the limitations of the invention involving anyone element or combinations of elements can be included in each aspectof the invention. This invention is not limited in its application tothe details of construction and the arrangement of components set forthor illustrated in the drawings. The invention is capable of otherembodiments and of being practiced or of being carried out in variousways.

Also, the phraseology and terminology used herein is for the purpose ofdescription and should not be regarded as limiting. The use of“including,” “comprising,” or “having,” “containing”, “involving”, andvariations thereof herein, is meant to encompass the items listedthereafter and equivalents thereof as well as additional items.

All references, patents and patent applications that are recited in thisapplication are incorporated by reference herein in their entirety.

We claim:
 1. A method for treating a renal failure subject, the methodcomprising: administering an effective amount of plasma gelsolin (pGSN)or cytoplasmic gelsolin (cGSN) to a renal failure subject in need ofsuch a treatment to raise the level of gelsolin in the subject above apredetermined value.
 2. The method of claim 1, wherein the subject isotherwise free of indications calling for treatment with gelsolin. 3.The method of claim 1, wherein the predetermined value is about 190ng/μl of plasma.
 4. The method of claim 1, wherein the predeterminedvalue is about 150 ng/μl of plasma.
 5. The method of claim 1, whereinthe predetermined value is about 120 ng/μl of plasma.
 6. The method ofclaim 1, wherein the subject is on dialysis.
 7. The method of claim 6,wherein the dialysis is hemodialysis or peritoneal dialysis.
 8. Themethod of claim 1, wherein the subject has end-stage renal disease. 9.The method of claim 1, wherein the gelsolin is administered orally,sublingually, buccally, intranasally, intravenously, intramuscularly,intrathecally, intraperitoneally, or subcutaneously.
 10. A method fortreating a renal failure subject having or at risk of developing aninfection, the method comprising: administering plasma gelsolin (pGSN)or cytoplasmic gelsolin (cGSN) to the renal failure subject, in aneffective amount to treat the infection or to reduce the risk of theinfection.
 11. The method of claim 10, wherein the infection is causedby a gram-positive bacterium, a gram-negative bacterium, an acid-fastbacillus, a spirochete, an actinomycete, a virus, a fungus, a parasite,Ureoplasma species, Mycoplasma species, Chlamydia species, orPneumocystis species.
 12. The method of claim 10, wherein the subject isotherwise free of indications calling for treatment with gelsolin. 13.The method of claim 10 further comprising administering a second agent.14. The method of claim 10, wherein the subject is on dialysis.
 15. Themethod of claim 14, wherein the dialysis is hemodialysis or peritonealdialysis.
 16. The method of claim 10, wherein the subject has end-stagerenal disease.
 17. The method of claim 10, wherein the gelsolin isadministered orally, sublingually, buccally, intranasally,intravenously, intramuscularly, intrathecally, intraperitoneally, orsubcutaneously.